Nucleoside analogs (NAs) - a mainstay of anticancer therapy - are prodrugs that upon conversion into their phosphorylated form mimic naturally occurring nucleotides. The clinically relevant NA metabolite is predominantly the triphosphorylated form, though some are active as the monophosphate. Importantly, the efficiency of prodrug-to-drug conversion can largely determine the clinical utility of NAs. For most NAs it is the initial conversion into the monophosphate form that is rate-limiting for their overall activation. The enzyme deoxycytidine kinase (dCK) is the most important kinase in terms of nucleoside analog activation. The physiological role of this nucleoside kinase is to convert dC, dA, and dG into their monophosphate form. This inherent promiscuity also enables NAs of various structures (e.g. purines, pyrimidines, with modifications in the sugar, base, or both moieties) to be accepted as substrates. Our goal is to decipher the molecular determinants of dCK substrate specificity and the requirements for efficient NA phosphorylation by this enzyme. This information will guide the engineering of dCK variants (dCKEN) that have unique and improved activities with select NAs. The long-term goal is to deliver dCKEN to cancer cells, where they will catalyze the activation of the NA to its cytotoxic form. In the initial funding period we solved the first ever dCK structure and revealed how 2'-substitutions in the sugar ring of pyrimidine NAs affect activity. In addition, we elucidated the structural basis for the lack of enantioselectivity by dCK. We will advance with structure/function studies of dCK to elucidate factors that influence the efficiency of nucleoside phosphorylation. For example, we will elucidate the mechanism behind the activation of dCK by post-translational phosphorylation of Ser74 (Aim 1). The structure/function studies will increase our understanding of the dCK enzymatic mechanism, its determinants of substrate specificity, and the conformational changes that occur during the reaction cycle. This garnered knowledge will be used to design dCK variants (i.e. dCKEN) that possess unique and improved catalytic activities (Aim 2). In Aim 3 we will test the potential of our dCKEN to sensitize various cancer-relevant cell lines to select NAs. In our therapeutic strategy we employ monoclonal antibodies (mAb) to deliver dCKEN in a specific manner to cancer cells. This is done by conjugating dCKEN to internalizing mAbs. After recognition of the cancer cells by the mAb portion of the conjugate, the molecule internalizes and delivers dCKEN into the cell. Subsequent NA administration will result in that NA activated (i.e. made cytotoxic) only in the targeted cells. Therefore, this strategy avoids many of the side effects that limit current cancer chemotherapeutics. Moreover, the modular nature of the conjugate means that diverse cancers could be treated (e.g. HER2+ breast cancer, CD33+ leukemias, PSMA+ prostate cancer), by simply conjugating the appropriate mAb to dCKEN